BACKGROUND: Colorectal cancer is still the second leading cause of cancer-related deaths and thus biomarkers allowing prediction of the resistance of patients to therapy and estimating their prognosis are needed. We designed a panel of 558 genes with pharmacogenomics records related to 5-fluorouracil resistance, genes important for sensitivity to other frequently used drugs, major oncodrivers, and actionable genes. We performed a target enrichment sequencing of DNA from tumors and matched blood samples of patients, and compared the results with patient prognosis stratified by systemic adjuvant chemotherapy. RESULTS: The median number of detected variants per tumor sample was 18.5 with 4 classified as having a high predicted functional effect and 14.5 moderate effect. APC, TP53, and KRAS were the most frequent mutated genes (64%, 59%, and 42% of mutated samples, respectively) followed by FAT4 (23%), FBXW7, and PIK3CA (16% for both). Patients with advanced stage III had more frequently APC, TP53, or KRAS mutations than those in stages I or II. KRAS mutation counts followed an increasing trend with grade (G1 < G2 < G3). The response to adjuvant therapy was worse in carriers of frameshift mutations in APC or 12D variant in KRAS, but none of these oncodrivers had prognostic value. Carriage of somatic mutations in any of the genes ABCA13, ANK2, COL7A1, NAV3, or UNC80 had prognostic relevance for worse overall survival (OS) of all patients. In contrast, mutations in FLG, GLI3, or UNC80 were prognostic in the same direction for patients untreated, and mutations in COL6A3, LRP1B, NAV3, RYR1, RYR3, TCHH, or TENM4 for patients treated with adjuvant therapy. The first association was externally validated. From all germline variants with high or moderate predicted functional effects (median 326 per patient), > 5% frequency and positive Manhattan plot based on 3-year RFS, rs72753407 in NFACS, rs34621071 in ERBB4, and rs2444274 in RIF1 were significantly associated with RFS, OS or both. CONCLUSIONS: The present study identified several putative somatic and germline genetic events with prognostic potential for colorectal cancer that should undergo functional characterization.

Biomedical Center Faculty of Medicine in Pilsen Charles University alej Svobody 1655 76 Pilsen 323 00 Czech Republic

Department of Oncology and Radiotherapeutics Faculty of Medicine and University Hospital in Pilsen Charles University Pilsen Czech Republic

Department of Surgery Faculty of Medicine and University Hospital in Pilsen Charles University Pilsen Czech Republic

Toxicogenomics Unit National Institute of Public Health Prague Czech Republic

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Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin [Internet]. 2021;71(3):209–49. 10.3322/caac.21660. 10.3322/caac.21660 PubMed DOI

SEER*Explorer. An interactive website for SEER cancer statistics [Internet] Surveillance Research Program, National Cancer Institute; Accessed January 24 2024 [https://seer.cancer.gov/explorer/].

Keum N, Giovannucci E. Global burden of colorectal cancer: emerging trends, risk factors and prevention strategies. Nat Rev Gastroenterol Hepatol [Internet]. 2019;16(12):713–32. 10.1038/s41575-019-0189-8. 10.1038/s41575-019-0189-8 PubMed DOI

Benson AB, Venook AP, Al-Hawary MM, Arain MA, Chen Y-J, Ciombor KK, et al. Colon cancer, version 2.2021, NCCN Clinical Practice guidelines in Oncology. J Natl Compr Canc Netw [Internet]. 2021;19(3):329–59. 10.6004/jnccn.2021.0012. 10.6004/jnccn.2021.0012 PubMed DOI

Heidelberger C, Chaudhuri NK, Danneberg P, Mooren D, Griesbach L, Duschinsky R, et al. Fluorinated pyrimidines, a new class of tumour-inhibitory compounds. Nat [Internet]. 1957;179(4561):663–6. 10.1038/179663a06.10.1038/179663a06 PubMed DOI

Wyatt MD, Wilson D, Wilson PM, Danenberg PV, Johnston PG, Lenz HJ, et al. Standing the test of time: targeting thymidylate biosynthesis in cancer therapy. Nat Rev Clin Oncol. 2009;66(5):282–98. 10.1038/nrclinonc.2014.51.10.1038/nrclinonc.2014.51 PubMed DOI

Wilson PM, Danenberg PV, Johnston PG, Lenz H-J, Ladner RD. Standing the test of time: targeting thymidylate biosynthesis in cancer therapy. Nat Rev Clin Oncol [Internet]. 2014;11(5):282–98. 10.1038/nrclinonc.2014.51. 10.1038/nrclinonc.2014.51 PubMed DOI

Pardini B, Kumar R, Naccarati A, Novotny J, Prasad RB, Forsti A, et al. J: 5-Fluorouracil-based chemotherapy for colorectal cancer and MTHFR/MTRR genotypes. Br J Clin Pharmacol. 2011;72(1):162–3. 10.1111/j.1365-2125.2010.03892.x. 10.1111/j.1365-2125.2010.03892.x PubMed DOI PMC

Carola C, Ghiringhelli F, Kim S, André T, Barlet J, Bengrine-Lefevre L, et al. FOLFIRI3-aflibercept in previously treated patients with metastatic colorectal cancer. World J Clin Oncol [Internet]. 2018;9(5):110–8. 10.5306/wjco.v9.i5.110. 10.5306/wjco.v9.i5.110 PubMed DOI PMC

Giacchetti S, Perpoint B, Zidani R, Le Bail N, Faggiuolo R, Focan C, et al. Phase III multicenter randomized trial of oxaliplatin added to chronomodulated fluorouracil-leucovorin as first-line treatment of metastatic colorectal cancer. J Clin Oncol [Internet]. 2000;18(1):136–47. 10.1200/JCO.2000.18.1.136. 10.1200/JCO.2000.18.1.136 PubMed DOI

Douillard JY, Cunningham D, Roth AD, Navarro M, James RD, Karasek P, et al. Irinotecan combined with fluorouracil compared with fluorouracil alone as first-line treatment for metastatic colorectal cancer: a multicentre randomised trial. Lancet [Internet]. 2000;355(9209):1041–7. 10.1016/s0140-6736(00)02034-1. 10.1016/s0140-6736(00)02034-1 PubMed DOI

Heczko L, Hlaváč V, Holý P, Dvořák P, Liška V, Vyčítal O, et al. Prognostic potential of whole exome sequencing in the clinical management of metachronous colorectal cancer liver metastases. Cancer Cell Int [Internet]. 2023;23(1):295. 10.1186/s12935-023-03135-x. 10.1186/s12935-023-03135-x PubMed DOI PMC

Hlaváč V, Červenková L, Šůsová S, Holý P, Liška V, Vyčítal O, et al. Exome sequencing of paired colorectal carcinomas and synchronous liver metastases for prognosis and therapy prediction. JCO Precis Oncol [Internet]. 2023;7:e2200557. 10.1200/PO.22.00557. 10.1200/PO.22.00557 PubMed DOI

Li H. Toward better understanding of artifacts in variant calling from high-coverage samples. Bioinformatics. 2014;30(20):2843–51. 10.1093/bioinformatics/btu356. 10.1093/bioinformatics/btu356 PubMed DOI PMC

Van der Auwera GA, Carneiro MO, Hartl C, Poplin R, Del Angel G, Levy-Moonshine A, et al. From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline. Curr Protoc Bioinf [Internet]. 2013;43(1):11. 10.1002/0471250953.bi1110s43.10.1002/0471250953.bi1110s43 PubMed DOI PMC

McLaren W, Gil L, Hunt SE, Riat HS, Ritchie GRS, Thormann A, et al. The ensembl variant effect predictor. Genome Biol [Internet]. 2016;17(1). 10.1186/s13059-016-0974-4. PubMed PMC

Mayakonda A, Lin D-C, Assenov Y, Plass C, Koeffler HP. Maftools: efficient and comprehensive analysis of somatic variants in cancer. Genome Res [Internet]. 2018;28(11):1747–56. 10.1101/gr.239244.118. 10.1101/gr.239244.118 PubMed DOI PMC

Gu Z, Eils R, Schlesner M. Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinf [Internet]. 2016;32(18):2847–9. 10.1093/bioinformatics/btw313.10.1093/bioinformatics/btw313 PubMed DOI

Talevich E, Shain AH, Botton T, Bastian BC, CNVkit. Genome-wide copy number detection and visualization from targeted DNA sequencing. PLoS Comput Biol [Internet]. 2016;12(4):e1004873. 10.1371/journal.pcbi.1004873. 10.1371/journal.pcbi.1004873 PubMed DOI PMC

Lai Z, Markovets A, Ahdesmaki M, Chapman B, Hofmann O, McEwen R, et al. VarDict: a novel and versatile variant caller for next-generation sequencing in cancer research. Nucleic Acids Res [Internet]. 2016;44(11):e108–108. 10.1093/nar/gkw227. 10.1093/nar/gkw227 PubMed DOI PMC

Han X, Zhang S, Zhou DC, Wang D, He X, Yuan D, et al. MSIsensor-ct: microsatellite instability detection using cfDNA sequencing data. Brief Bioinform [Internet]. 2021;22(5). 10.1093/bib/bbaa402. PubMed PMC

Nowak JA, Yurgelun MB, Bruce JL, Rojas-Rudilla V, Hall DL, Shivdasani P, et al. Detection of mismatch repair deficiency and microsatellite instability in colorectal adenocarcinoma by targeted next-generation sequencing. J Mol Diagn [Internet]. 2017;19(1):84–91. 10.1016/j.jmoldx.2016.07.010. 10.1016/j.jmoldx.2016.07.010 PubMed DOI PMC

Albayrak A, Garrido-Castro AC, Giannakis M, Umeton R, Manam MD, Stover EH, et al. Clinical pan-cancer assessment of mismatch repair deficiency using tumor-only, targeted next-generation sequencing. JCO Precis Oncol [Internet]. 2020;4(4):1084–97. 10.1200/PO.20.00185. 10.1200/PO.20.00185 PubMed DOI PMC

Goldman MJ, Craft B, Hastie M, Repečka K, McDade F, Kamath A, et al. Visualizing and interpreting cancer genomics data via the Xena platform. Nat Biotechnol [Internet]. 2020;38(6):675–8. 10.1038/s41587-020-0546-8. 10.1038/s41587-020-0546-8 PubMed DOI PMC

Sargent DJ, Wieand HS, Haller DG, Gray R, Benedetti JK, Buyse M, Labianca R, Seitz JF, O’Callaghan CJ, Francini G, et al. Disease-free survival versus overall survival as a primary end point for adjuvant colon cancer studies: individual patient data from 20,898 patients on 18 randomized trials. J Clin Oncol. 2005;23(34):8664–70. 10.1200/JCO.2005.01.6071. 10.1200/JCO.2005.01.6071 PubMed DOI

Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Roy Stat Soc B Met. 1995;57:289–300.10.1111/j.2517-6161.1995.tb02031.x DOI

Innocenti F, Mu W, Qu X, Ou F-S, Kabbarah O, Blanke CD, et al. DNA mutational profiling in patients with colorectal cancer treated with standard of care reveals differences in outcome and racial distribution of mutations. J Clin Oncol [Internet]. 2023;JCO2300825. 10.1200/JCO.23.00825. PubMed PMC

Li Y, Pan C, Gao Y, Zhang L, Ji D, Cui X, et al. Total neoadjuvant therapy with PD-1 blockade for high-risk proficient mismatch repair rectal cancer. JAMA Surg [Internet]. 2024. 10.1001/jamasurg.2023.7996. 10.1001/jamasurg.2023.7996 PubMed DOI PMC

Baxter NN, Kennedy EB, Bergsland E, Berlin J, George TJ, Gill S, Gold PJ, Hantel A, Jones L, Lieu C, et al. Adjuvant therapy for stage II Colon cancer: ASCO Guideline Update. J Clin Oncol. 2022;40(8):892–910. 10.1200/JCO.21.02538. 10.1200/JCO.21.02538 PubMed DOI

Datta J, Bianchi A, De Castro Silva I, Deshpande NU, Cao LL, Mehra S, et al. Distinct mechanisms of innate and adaptive immune regulation underlie poor oncologic outcomes associated with KRAS-TP53 co-alteration in pancreatic cancer. Oncogene [Internet]. 2022;41(28):3640–54. 10.1038/s41388-022-02368-w. 10.1038/s41388-022-02368-w PubMed DOI

Gu M, Xu T, Chang P. KRAS/LKB1 and KRAS/TP53 co-mutations create divergent immune signatures in lung adenocarcinomas. Ther Adv Med Oncol [Internet]. 2021;13:17588359211006950. 10.1177/17588359211006950. 10.1177/17588359211006950 PubMed DOI PMC